Cyclic AMP-dependent protein kinases play an important role in normal cell differentiation and cell replication. Many hormones/chemical substances affect cell metabolism and gene expression by regulating the level of intracellular cyclic AMP (cAMP), which in turn regulates the activity of cAMP-dependent protein kinases. The tetrameric holoenzyme of cAMP-dependent protein kinase consists of two identical regulatory (R) and catalytic (C) subunits. The catalytic subunits dissociate from the holoenzyme on the binding of two molecules of cAMP to each of the regulatory subunits. The free activated catalytic subunit phosphorylates serine and threonine residues on proteins and thereby mediates the biological response to the hormonal stimulus. It has further been shown that the free regulatory subunit may have a direct effect on the regulation of cellular functions independent of phosphorylation (Lohmann et al., 1984).
Originally two major types of cAMP-dependent protein kinases (types I and II) were described (Corbin et al., 1975). This classification was based on the sequential elution profile from DEAE-cellulose columns using increasing salt concentrations. These kinases were shown to be distinguished primarily by their regulatory subunits (RI and RII), which differ in their molecular weights, affinities for cAMP and cAMP analogues (Corbin et al., 1982; Robinson-Steiner and Corbin 1983), antigenicity (Fleischer et al., 1976; Kapoor et al., 1979; Lohmann et al., 1983) their ability to be autophosphorylated (Corbin et al., 1975; Hofmann et al., 1975; Rosen et al., 1975) and their amino acid sequences (Takio et al., 1984; Titani et al., 1984).
Recent studies, primarily involving cDNA cloning and sequencing, show a multiplicity in R and C subunit forms. Four different regulatory subunits (RI.alpha., RI.beta., RII.sub.51 (RII.beta.), and RII.sub.54 (RII.alpha.)) and two different catalytic subunits (C.alpha. and C.beta.) for cAMP-dependent protein kinases have now been identified at the gene/mRNA level. None of these have yet been characterized in human material at the gene/mRNA level. The bovine skeletal muscle type of RI (Titani et al., 1984), with an apparent molecular mass of 49 kD and found in most tissues, should now be designated RI.alpha.. Complementary DNA clones for this protein have been characterized (Lee et al., 1983). A second isoform of RI, (designated RI.beta.), which is only expressed in brain and germinal cells, has recently been identified by cDNA cloning from a mouse cDNA library (Dr. G. S. McKnight, unpublished data). For some time, two forms of RII have been recognized. A 54 kD form (rat) present in most tissues (Jahnsen et al., 1985; 1986a), has been designated RII5 4 (RII.alpha.), while a 51 kD form found in brain, granulosa cells, testis and adrenal tissue (Jahnsen et al., 1986a; 1986b) has been called RII.sub.51 (RII.beta.). A partial cDNA clone for RII.sub.51 from rat granulosa cells has been described (Jahnsen et al., 1986b), and recently the cloning of RII.sub.54 from rat skeletal muscle and mouse brain has been reported (Scott et al., 1987). To further complicate the picture, two different gene products for the C subunit have been revealed in mouse and bovine tissues (Uhler et al., 1986a; 1986b; Showers et al., 1986). These have been designated C.alpha. and C.beta.. In most tissues examined C.alpha. mRNA is more abundant than C.beta. mRNA.
A well defined domain structure appears to be retained in each R-subunit. The protein can be divided into thirds based on the amino acid sequence. The NH.sub.2 -terminal third of the molecule contains an essential recognition site for the C-subunit, and is also the major site of interaction between the two protomers of the R.sub.2 dimer. The COOH-terminal two-thirds of the molecule contain repeating segments which represent the two cAMP-binding sites.
Cyclic AMP-dependent protein kinases have previously been reported to play a central role in connection with human diseases such as cystic fibrosis (Frizzell et al., 1986: Welsh and Lietke, 1986), cancer (e.g. breast cancer (Cho-Chung, 1985)) and skin diseases (e.g. psoriasis (Brion et al., 1986)). In these cases the protein products of the cAMP-dependent protein kinase genes have been studied. The methods which have been used in these protein studies have mainly been DEAE cellulose chromatography, assays of protein kinase activity, photoaffinity labelling using (.sup.32 P) azido-cAMP and immunological methods. The sensitivity and specificity of these methods of measurement make it difficult/impossible to distinguish the various gene products for cAMP-dependent protein kinases. Modern methods using recombinant DNA technology have enabled the applicants to identify and separate different subunits of cAMP-dependent protein kinases. These studies have been performed using bovine, rat and mouse material.